Fibroblast activation protein (FAP) was originally identified as a serine protease on reactive stromal fibroblasts [1, 2]. Subsequent molecular cloning revealed that FAP is identical to seprase, a 170 kDa membrane associated gelatinase that is expressed by melanoma cell lines [3, 4]. Full length cDNA encoded a type H transmembrane protease of 760 amino acids (aa) highly homologous to dipeptidyl peptidase IV (DPPIV) with a 52% aa identity over the entire sequence and almost 70% identity in the catalytic domain [3, 5]. U.S. Pat. No. 5,587,299, incorporated herein by reference, describes nucleic acid molecules encoding FAP and applications thereof.
FAP and DPPIV have similar gene sizes and are chromosomally adjacent to each other at 2q24, suggesting a gene duplication event (Genebank accession number U09278). Both proteins are members of the prolyl peptidase family [1, 6]. This class of enzymes is inducible, active on the cell surface or in extracellular fluids, and uniquely capable of cleaving N-terminal dipeptides from polypeptides with proline or alanine in the penultimate position [7]. DPPIV, also termed CD26, is constitutively expressed by several cell types including fibroblasts, endothelial and epithelial cells, leukocyte subsets like NK-cells, T-lymphocytes and macrophages. A small proportion of DPPIV circulates as soluble protein in the blood. In contrast to DPPIV, FAP is typically not expressed in normal adult tissue [1] and its proteolytically active soluble form is termed a2-Antiplasmin Cleaving Enzyme (APCE) [8]. Marked FAP expression occurs in conditions associated with activated stroma, including wound healing, epithelial cancers, osteoarthritis, rheumatoid arthritis, cirrhosis and pulmonary fibrosis [4, 9-11].
The FAP structure has been solved (PDB ID 1Z68) and is very similar to that of DPPIV [12]. FAP is anchored in the plasma membrane by an uncleaved signal sequence of approximately 20 amino acids and has a short, amino terminal, cytoplasmic domain of six amino acids [3-5]. The major part of the protein, including the catalytic domain, is exposed to the extracellular environment [13]. The FAP glycoprotein is a homodimer consisting of two identical 97-kDa subunits. Each FAP-monomer subunit consists of two domains, an αβ hydrolase domain (aa 27-53 and 493-760) and an eight-blade β propeller domain (aa 54-492) that enclose a large cavity. A small pocket within this cavity at the interface of both domains contains the catalytic triad (Ser624, Asp702 and His734) [12]. FAP gains its enzymatic activity upon homodimerization of the subunits [14] and beside its dipeptidyl peptidase activity, FAP also has collagen type I specific gelatinase [15] and endopeptidase activity [16]. The β propeller acts as scaffolding for protein-protein interactions and determines substrate and extracellular matrix (ECM) binding [17]. Furthermore, the β propeller is involved in forming supra-molecular complexes of FAP with other prolyl peptidases or with other membrane-bound molecules [18, 19]. The formation of heteromeric or tetrameric complexes of FAP and DPPIV were found to be associated with invadopodia of migrating cells on a collagen substrate [20]. Type I collagen induces a close association of FAP with β1 integrins, thereby playing major organizational roles in the formation and adhesion of invadopodia [21]. Although the involved mechanisms are not understood in detail, the formation of such proteinase-rich membrane domains at the cellular invasion front contributes to directed pericellular ECM degradation [22]. This indicates that FAP and ECM interactions may be closely related to invasive cell behaviour by influencing cell adhesion, migration, proliferation and apoptosis through integrin pathways [19, 21, 23] and supports o role of FAP in disease pathogenesis and progression [24]. In summary, FAP is recognized as a multifunctional protein that executes its biological functions in a cell dependent manner through a combination of its protease activity and its ability to form complexes with other cell-surface molecules. Over-expression of FAP in epithelial and fibroblastic cell lines promotes malignant behaviour [22], pointing to the clinical situation, where cellular expression levels of FAP are correlated with worse clinical outcome [25, 26].
Through paracrine signaling molecules, cancer cells activate stromal fibroblasts and induce the expression of FAP, which in turn, affects the proliferation, invasion and migration of the cancer cells. Recent studies have demonstrated that TGF-β is the dominant factor in promoting FAP protein expression (Chen, H et al (2009) Exp and Molec Pathology, doi: 10.1016/j.yexmp. 2009.09.001). FAP is heavily expressed on reactive stromal fibroblasts in 90% of human epithelial carcinomas, including those of the breast, lung, colorectum and ovary (Garin-Chesa, P et al (1990) PNAS USA 87: 7236-7239). Chen et al have recently shown that FAPα influences the invasion, proliferation and migration of HO-8910PM ovarian cancer cells (Chen, H et al (2009) Exp and Molec Pathology, doi: 10.1016/j.yexmp. 2009.09.001).
The morphological and functional properties of FAP promote the investigation of FAP as a therapeutic target. The disease related and cell surface bound expression pattern especially qualifies FAP for antibody targeting. With regard to the pathophysiological involvement in ECM remodelling, targeting strategies should aim at the disruption of the signalling supra-molecular FAP complexes. Although FAP has attracted increased interest as a target for antibody based immunotherapy, data of therapeutically active native FAP-specific antibodies are missing to date. The monoclonal antibody F19 was the first antibody investigated in a phase I clinical trial targeting metastatic colorectal cancer [30]. This trial served as a proof of principle for anti-FAP based tumor stroma targeting [1]. Although patients included in the trial had extensive scarring due to surgery, no specific enrichment of 131I-F19 could be detected at these sites. There were no toxic side effects associated with intravenous administration of iodine131 labelled F19 and carcinoma lesions were specifically detected by imaging down to a size of 1 cm in diameter. With regard to the immunogenicity of murine antibodies in humans, recent phase I and phase II clinical trials were conducted using the humanized version of F19, called Sibrotuzumab [31, 32]. Results from these trials demonstrated the safe and well tolerated administration of Sibrotuzumab. Similar to the results obtained in the pivotal phase I trial [30], trace-labelling with 131I and imaging analysis revealed the specific accumulation of Sibrotuzumab at the tumor area. Unfortunately, unconjugated Sibrotuzumab demonstrated no anti-tumor or any therapeutic activity, respectively [32]. Although the biologic function of FAP is still not known in detail, its dipeptidyl peptidase activity was postulated to be involved in tumor progression and metastasis [15, 33]. The lack of Sibrotuzumab to affect FAP enzymatic function was suggested to be the reason for the lack of therapeutic efficacy [34]. In consequence, anti-FAP directed polyclonal antibodies have been raised in order to inhibit the catalytic activity in-vitro. Indeed, treatment of FAP-positive xenografts with anti-FAP anti-sera attenuated tumor growth [13]. However, since polyclonal sera were raised by immunization of rabbits with murine FAP, it is most likely that additional epitopes, different from the catalytic domain, have also been targeted. Therefore, it is difficult to conclude from this study that anti-tumor effects seen really depended on dipeptidyl-peptidase inhibition.
Thus, while the extant evidence of activity of FAP antibodies is encouraging, the observed limitations on efficacy and anti-tumor activity remain. Accordingly, it would be desirable to develop FAP antibodies, particularly antibodies which can be utilized in mouse animal models and which demonstrate increased efficacy and applicability in diagnosis and therapy, and it is toward the achievement of that objective that the present invention is directed.
The citation of references herein shall not be construed as an admission that such is prior art to the present invention.